1. Field of the Invention
The present invention relates to a test for the detection of exogenous fever-producing substances in which an incubation of the substance with human monocytes and lymphocytes produces human leukocytic pyrogen.
2. Description of the Prior Art
The recent development of the ability to manipulate genes has resulted in a new source of pharmaceutical substances. Genes for desirable, naturally occurring drugs, such as interferon, insulin, and human growth hormone, have been removed from normal human cells and inserted into bacterial cells, which may then be used as biological factories of these important biochemical substances. It is anticipated that more and more pharmaceuticals will become available in this fashion as techniques of genetic manipulation continue to improve.
Unfortunately, pharmaceuticals derived from bacteria are often contaminated with minute quantities of material that may cause undesirable effects when administered parenterally to humans. One such undesirable effect is the production of fever. Studies of fever in response to injections go back to the 19th century, when the lipopolysaccharide of gram-negative organisms (bacterial endotoxin) was recognized as a fever-producing agent (a pyrogen). This is only one of many substances, most of microbial origin, that cause fever when injected into humans. Although other animals may also demonstrate fever when injected with the same substances, there is considerable variation from species to species, and the production of fever by a substance in one species may not mean that fever will be produced in another. Thus tests for pyrogenicity of materials intended for human parenteral administration may be misleading if based on the reaction of another species.
The only standard test currently available for the detection of the pyrogenicity of materials are the rabbit pyrogen test (USP) and the Limulus amebocyte lysate test. As these tests are based on the response of a non-human animal or cells derived from a non-human animal, their reliability has often been questioned, for example, in "Nonspecificity of the Limulus amebocyte lysate test: Positive reactions with polynucleotides and proteins," Elin et al., J. Inf. Dis., 128:349-352 (1973).
Because of the shortcomings of animal-based tests, a test more closely corresponding to human fever response is needed, one which will mimic in vitro the fever response that exogenous pyrogens induce when they are administered parenterally to humans. Investigations into the pathogenesis of fever production by pyrogens has established that pyrogens do not cause fever by a direct effect on the thermoregulatory center but rather by an indirect mechanism. This mechanism involves the stimulation of leukocytes by exogenously injected pyrogens to synthesize and release an endogenous pyrogen, which is also called leukocytic pyrogen (LP). LP is a polypeptide of 15,000 daltons which is produced by phagocytic leukocytes, primarily the mononuclear phagocytes (monocytes). Evidence that LP mediates fever is drawn from 30 years of research into the pathogensis of fever. Following the injection of exogenous pyrogens obtained from either microbial or non-microbial sources, LP appears in the circulation of experimental animals. In addition, incubating exogenous pyrogens with phagocytic leukocytes in vitro induces the production and release of LP into the supernatant medium. Human leukocytes which have been stimulated by exogenous pyrogens in vitro often release LP into the culture medium after several hours of incubation. When the medium is injected into human subjects, there is rapid onset of fever. An assay for the presence of human LP can also be carried out in rabbits and in mice, where it produces a similar febrile response. The rapid onset of fever is characteristic of LP from all species studied and represents the ability of this molecule to directly affect the thermoregulatory center and initiate fever.
The critical role of LP in the pathogenesis of fever is underscored by the correlation that exogenous pyrogens which produce fever in humans also stimulate human leukocytes to release LP in vitro. This has been shown for a variety of exogenous pyrogens including several microbial pyrogens, poly I:C, bleomycin, colchicine, bacterial endotoxin, etiocholanolone, and synthetic adjuvants [reviewed in Dinarello and Wolff, Seminars in Inf. Dis., 2:173-192 (1979)]. Of particular interest are studies of etiocholanolone, which is non-pyrogenic for the monkey, rabbit, dog, cat, guinea pig, rat or mouse but highly pyrogenic when injected into humans. When incubated with leukocytes, etiocholanolone induces LP production from only human cells. Additionally, the synthetic adjuvant muramyl dipeptide and several of its analogs produce fever in humans and cause LP production from human monocytes in vitro; moreover, side-chain substitution of these adjuvants associated with decreased ability to stimulate LP production in vitro is also less pyrogenic for humans. Therefore, the ability of a substance to induce LP production in vitro from human leukocytes seems to correlate with its ability to cause fever in the intact host.
There are, however, no established procedures for the certain and unequivocal detection of exogenous pyrogens using a test based on induction of LP synthesis. Although many investigations of the pathogenesis of fever production have involved measurement of LP production by pyrogens during in vitro incubations with leukocytes, not all pyrogenic substances induce LP production during a normal period of incubation, which is generally 24 hours. Although longer incubations may be carried out in an attempt to induce LP production, such longer incubations using these prior art procedures invariably lead to autostimulation, which produces LP even in the absence of any added material. Thus, prior art methods for the detection of LP production lead to false negatives if the time of incubation is about 24 hours or less and false positives if the time of incubation is much more than 24 hours.
Thus, at the time the present invention was made, there was still a need for an accurate and reliable test for determining which of those substances intended for parenteral administration to humans were either pyrogens themselves or were contaminated with pyrogens.